A new fluorometric assay for the study of DNA-binding and 3′-processing activities of retroviral integrases and its use for screening of HIV-1 integrase inhibitors

Andrey Anisenko, Julia Agapkina, Timofei Zatsepin, Dmitry Yanvarev, Marina Gottikh

Research output: Contribution to journalArticlepeer-review

8 Citations (Scopus)

Abstract

Fluorometry using a substrate DNA labeled with a single fluorophore (6-carboxyfluorescein) at the 3′-end of the processed strand was shown to be a useful tool for monitoring DNA-binding and 3′-processing activities of HIV-1 and PFV integrases (INs). The DNA binding to either of the INs resulted in a fluorescence signal decrease, which is likely due to the fluorescence quenching by aromatic amino acids located near the 3′-end of the processed strand. The fluorescence deviations upon the 3′-processing strongly depended on the sequence of the fluorescein-labeled terminus of the substrate DNA. In the case of HIV-1 IN, a time-dependent fluorescence decrease was detected. Since it correlated with the rate of 3′-processing resulted in the labeled GT dinucleotide accumulation, it might be explained by the fluorescein quenching by a guanosine residue in the single-stranded dinucleotide. The 3′-processing catalyzed by PFV IN led to the fluorescence enhancement. We ascribed it to the migration of the cleaved AT dinucleotide conjugated with fluorescein away from the amino acids that could quench its fluorescence. The fluorescence-based assay was used for the search of new HIV-1 IN inhibitors. Some bisphosphonate derivatives, which are known to block the phosphorolytic activity of HIV-1 reverse transcriptase, were shown to inhibit HIV-1 IN at micromolar concentrations. This property makes bisphosphonates promising agents for the development of HIV-1 inhibitors affecting two viral enzymes.

Original languageEnglish
Pages (from-to)2382-2390
Number of pages9
JournalBiochimie
Volume94
Issue number11
DOIs
Publication statusPublished - Nov 2012
Externally publishedYes

Keywords

  • 3′-end fluorescein-labeled oligonucleotides
  • 3′-Processing inhibitors
  • Fluorescence-base assay
  • HIV-1 integrase

Fingerprint

Dive into the research topics of 'A new fluorometric assay for the study of DNA-binding and 3′-processing activities of retroviral integrases and its use for screening of HIV-1 integrase inhibitors'. Together they form a unique fingerprint.

Cite this